The principal objective of this work is to design isozyme-specific activator/inhibitors of protein kinase C (PK-C) capable of binding selectively to the regulatory C1 domain of PK-C isozymes, as well as to other enzymes with structurally similar C1 domains. These include families of proteins involved in signal transduction, namely PKD, the chimaerins and RasGRP, all of which show strong binding affinity for the phorbol esters. The main synthetic effort has been to design simple and chemically accessible compounds that are structurally related to the natural second messenger diacylglycerol (DAG), and yet have binding potencies similar to those shown by the phorbol esters. The expansion of a library of compounds based on a 4,4-disubstituted-gamma butyrolactone template as a conformationally rigid DAG scaffold (DAG-lactones) continues in order to reach the best possible combination of alkyl and acyl chains capable of improving hydrophobic interactions with a group of highly conserved hydrophobic amino acids along the rim of the C1 domain (e.g., the Phe-243, Leu-250, Trp-252 and Leu-254 in PK-Cdelta). One of the most potent compounds yet discovered, (Z)-{1-(Hydroxymethyl)-4-[4-methyl-3 (methylethyl)pentylidene]-3-oxo-2-oxolanyl}methyl 4-methyl-3-(methylethyl)pentanoate, displayed low namomolar (2 nM) binding affinity for PK-C and was shown to selectively translocate PK-Cdelta to the Golghi apparatus inside the cell (J. Biol. Chem. 1999, 274, 37233 37239). This compound also showed potent apoptotic activity in prostate cancer cells and displayed selective antitumor activity in the NCI 60-cell line in vitro screen against breast, colon, and leukemia cell lines at low nanomolar concentrations. The incorporation of a hydroxamide side chain to our main DAG-lactone template showed excellent binding affinity (5 nM) for PK-C. According to our docking simulations with the X-ray crystal structure of the C1 domain of PK-C delta bound to phorbol 13-O-acetate, the NH-OH pharmacophore binds at the same site on PK-C delta that the 4-beta-OH of the phorbol esters binds to. This fourth pharmacophore has helped define the binding mode of these molecules by eliminating the ambiguity of two energetically equivalent binding modes observed with compounds possessing a three pharmacophore set. A similar combinatorial approach to the one used for the DAG-lactones was applied to the design a DAG analogue that combined the highest binding affinity with the lowest lipophilicity (log P). The best DAG analogue identified by this study has a binding affinity of 38 nM and a log P of 3.8. The combination of these parameters makes this compound equivalent to the potent dioctanoylgycerol, and yet it is 1.4 orders of magnitude more hydrophilic. Relative to the commonly used OAG (affinity = 50 nM), the newly discovered compound is 1.3 times more potent and 1.8 orders of magnitude less hydrophobic. The consequences of these dramatic changes are being investigated in systems where the lipid requirement for binding is less important. Protein kinase C isozymes. Chimaerins. Activation/inhibition, Drug design. Zinc finger. Apoptosis, cellular localization.